The normal human breast cell line Hs 578Bst and the human breast cancer cell line MDA-MB-231 were purchased from the American Type Tissue Culture Collection (Manassas, VA, USA) and cultured in Hyclone™ Dulbecco's modified Eagle's medium (DMEM)/F12 (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone™; GE Healthcare Life Sciences), 100 U/ml penicillin and streptomycin, in 25 cm² culture flasks at 37°C in a humidified atmosphere with 5% CO₂. Following serum starvation, cells were preincubated with either 100 mM Shh/cyclopamine (Sigma-Aldrich China, Inc., Shanghai, China) or vehicle control [0.1% dimethyl sulfoxide (DMSO)] for 48 h.

Cell viability was determined by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). In order to determine the impacts of Shh signaling on the human breast cancer cell line, MDA-MB-231, cells were cultured to ~70% confluence then starved in serum-free DMEM (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) overnight. The cells were then preincubated with Shh/cyclopamine for 48 h, cultured in fresh medium including 0.5 mg/ml MTT for 4 h, then DMSO was added to the wells to dissolve the blue formazan products and the density was determined spectrophotometrically at a wavelength of 550 nm using a microplate reader. Densitometric analysis was performed in which the level of in Shh-treated cells was normalized against the level in the DMSO group, which was arbitrarily set at 1. Each experiment was independently performed at least three times.

Cells were treated with either Shh/Cyclopamine or DMSO as described above. Cells were stained with 25 mg/l ethidium bromide (Sangon Biotech Co., Ltd., Shanghai, China) and the apoptotic status of samples was analyzed by a flow cytometer at an excitation wavelength of 488 nm.

Total RNA was isolated from cultured cells with RNAiso Plus (Takara Bio, Inc., Otsu, Japan) following the manufacturer's instructions. Routine DNase (Applied Biosystems; Thermo Fisher Scientific, Inc.) treatment (1 unit DNase I/mg total RNA) was performed prior to RT. A 10 ng aliquot of total RNA was reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) with specific primers for SHH, PTCH, SMO, GLI1, snail family zinc finger 2 (SLUG), B-cell lymphoma 2 (BCL-2), cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer (Sangon Biotech Co., Ltd.) sequences were as follows: Forward, 5′-TCCTCGCTGCTGGTATG-3′ and reverse, 5′-AAGCGTTCAACTTGTCCTTA-3′ for SHH; forward, 5′-AAATTCAAACCCTCCCTCTG- 3′ and reverse, 5′-AAGAGTCTCTGAAACTTCGC-3′ for PTCH; forward 5′-CTACAACGTGTGCCTGG-3′ and reverse, 5′-GGTCATTCTCACACTTGGG-3′ for SMO; forward, 5′-CAGCGCCCAGACAGA-3′ and reverse, 5′-CGGACATGAGGTTAGCTTG-3′ for GLI1; forward, 5′-ACACATTAGAACTCACACGG-3′ and reverse, 5′-AAGCACTATGTCACAACTTCA-3′ for SLUG; BCL2, forward, 5′-GTGGGAGCTTGCATCAC-3′ and reverse, 5′-ATTTCTACTGCTTTAGTGAACCT-3′ for BCL-2; forward, 5′-TCTATAAATTGAGCCCGCAG-3′ and reverse, 5′-TACCAGAGTTAAAAGCAGCC-3′ for GAPDH. All primers were used at a concentration of 10 µM. qPCR amplifications were performed in reaction volumes of 20 µl containing 10 µl TaqMan 2X Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems; Thermo Fisher Scientific, Inc.), 1 µl 20X TaqMan RNA Assay mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and 1.33 µl template cDNA. The thermal cycling conditions were a hot-start step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. PCR reactions were conducted on a Bio-Rad iQ5 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Relative mRNA expression was normalized against the endogenous control, GAPDH, using the comparative quantification cycle (Cq) method (2^−ΔΔCq) (17). Reactions were repeated in triplicate and Bio-Rad CFX Manager Software 1.6 (Bio-Rad Laboratories) was used for quantitative analysis of mRNA expression.

For total protein extraction, the cells were treated with radioimmunoprecipitation assay buffer [Beijing Solarbio Science & Technology Co., Ltd., Beijing, China; 50 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate (SDS)] containing 1% (v/v) phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.), 0.3% (v/v) protease inhibitor (Sigma-Aldrich) and 0.1% (v/v) phosphorylated proteinase inhibitor (Sigma-Aldrich). The lysates were centrifuged at 12,000 rpm (13,400 × g) at 4°C for 15 min, and the relative concentration of total proteins in the supernatant was quantified using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Aliquots containing equal amounts of protein (15 µg) were separated by SDS-polyacrylamide gel electrophoresis [10% (v/v)] and transferred onto a polyvinylidene difluoride (PVDF) membrane at 300 mA for 2 h. To block nonspecific binding, the PVDF membrane was incubated in 8% (w/v) milk in Tris-buffered saline with Tween-20 for 2 h at room temperature. The membranes were then incubated overnight at 4°C with the following primary antibodies: Polyclonal rabbit anti-GAPDH (dilution, 1:2,000; #sc-25778), polyclonal rabbit anti-Shh (dilution,1:1,000; #sc-9024) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), polyclonal rabbit anti-Ptch (dilution, 1:1,000; #A01; Abnova Corporation, Taiwan, China), polyclonal rabbit anti-Smo (dilution, 1:1,500; #sc-13943), polyclonal rabbit anti-Gli1 (dilution, 1:1,000; #sc-20687), polyclonal rabbit anti-SLUG (dilution, 1:1,000; #sc-20687), polyclonal rabbit anti-Bcl-2 (dilution, 1:2,000; #sc-492) (Santa Cruz Biotechnology, Inc.), and monoclonal rabbit anti-Cyclin D1 (dilution, 1:1,000; #2978; Cell Signaling Technology, Danvers, MA, USA). The following day, the membranes were washed four times (5 min each) with phosphate-buffered saline with Tween-20, then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Abmart, Arlington, MA, USA; dilution, 1:5,000) for 2 h at room temperature. Following incubation, the membranes were washed four times (5 min each) and the relative protein level was determined using enhanced chemiluminescence (EMD Millipore, Bedford, MA, USA) with the assistance of the ChemiDoc™ Touch Imaging system (Bio-Rad Laboratories, Inc.); each target protein was normalized against GAPDH.

Cells were grown as a confluent monolayer in 6-well plates. To initiate migration, the cell layer was scratched using a pipette tip. Time-lapse images of cell morphology were captured at 24 and 48 h using a fluorescence stereomicroscope (#MZ16FA; Leica Microsystems, Beijing, China). The migration abilities were quantified by measuring the area of the scratched regions using Image-Pro Plus version 4.5 software (Media Cybernetics, Inc., Rockville, MD, USA). The experiment was performed three times.

Following pretreatment with Shh/cyclopamine for 48 h, the culture medium was aspirated, centrifuged at 3,000 rpm (1,006.2 × g) for 20 min, and the levels of matrix metalloproteinase (MMP)-2 and −9 in the supernatant were assayed using human MMP-2 and −9 ELISA kits (Beijing Rui'er Xinde Technology, Beijing, China) according to the manufacturer's instructions.

All data are expressed as the mean ± standard error of the mean. The number of independent experiments is represented by ‘n.’ Multiple comparisons were performed using one-way analysis of variance followed by Tukey's multiple-comparisons test. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of Shh-Gli signaling components were determined in the human breast cancer cell line MDA-MB-231 using western blotting. The Shh-Gli signaling receptors Ptch and Smo, and the target transcription factors Gli1 and Gli2, were found to be highly expressed at the protein level in MDA-MB-231 cells compared with the normal human breast cell line Hs 578Bst (Fig. 1A). Meanwhile, the relative mRNA levels of PTCH, SMO and GLI were also markedly higher in MDA-MB-231 cells relative to Hs 578Bst cells, as determined by RT-qPCR (P<0.01; Fig. 1B). Together, these results indicate that components of Shh-Gli signaling are overexpressed in the human breast cancer cell line MDA-MB-231, as are the target transcripts of this signaling (PTCH, GLI1, and GLI2). This suggests that Shh-Gli signaling is activated in MDA-MB-231 cells.

In various types of cancers, Shh-Gli signaling has been reported to be widely activated (18). Exogenous activation of Shh signaling also significantly stimulates cell proliferation (11). Cyclopamine, a specific inhibitor of the Shh-Gli signaling pathway, decreases Shh-induced malignant cancer cell proliferation (19,20). To determine the effects of Shh-Gli signaling on cell proliferation, MDA-MB-231 cells were treated with exogenous Shh, cyclopamine or exogenous Shh plus cyclopamine for 24, 48, or 72 h, and cell viability was determined using an MTT assay. In the presence of exogenous Shh, MDA-MB-231 cell viability was significantly enhanced, while suppression of Shh-Gli signaling with cyclopamine reduced cell viability in a time-dependent manner (P<0.01; Fig. 2A). This result indicates that Shh-Gli signaling regulates human breast cancer cell proliferation. Furthermore, Gli is considered as a hallmark of the Shh-Gli signaling pathway (21). Thus, the transcript level of Gli was determined using RT-qPCR. Compared with the control group, treatment with exogenous Shh significantly increased Gli mRNA levels, while cyclopamine markedly reduced Gli transcription (Fig. 2B). Together, these results indicate that Shh-Gli signaling is important in breast cancer cell proliferation.

To further explore the effects of Gli on cell proliferation, the protein levels of Bcl-2 and cyclin D1 were analyzed using western blotting. In the presence of Shh, Bcl-2 and cyclin D1 were significantly enhanced (Fig. 3A). By contrast, when Gli expression was inhibited with cyclopamine, Bcl-2 and cyclin D1 were remarkably reduced. Thus Gli may regulate the protein levels of Bcl-2 and cyclin D1 in MDA-MB-231 cells. Meanwhile, cyclopamine treatment significantly reduced Gli, Bcl-2 and cyclin D1 protein levels induced by Shh. Furthermore, cyclopamine significantly enhanced cell apoptosis, while Shh treatment reversed this increase in apoptotic rate (Fig. 3B). These data suggested that overexpression of Gli positively regulates transcription of Bcl-2 and cyclin D1, thereby regulating MDA-MB-231 cell proliferation and survival.

In the tumor progression process, EMT is important in tumor motility and invasion (22). E-cadherin is a hallmark molecule of the EMT process, and its downregulation indicates enhanced cell motility (23). SLUG is a downstream target of Shh-Gli signaling, and is suggested to serve key roles in the EMT process (13). In the current study, western blotting demonstrated that 72 h treatment of MDA-MB-231 cells with human sonic hedgehog, n-terminus (N-Shh) significantly reduces E-cadherin protein levels (Fig. 4A). By contrast, cells treated for 72 h with cyclopamine exhibited enhanced E-cadherin levels, whereas the expression of SLUG was significantly suppressed. These changes were completely restored by pretreatment with cyclopamine prior to N-Shh stimulation (Fig. 4A).

Subsequently, Shh-mediated cell migration and invasion were evaluated to determine whether these processes involved elevated levels of MMPs, which are associated with tumor invasiveness (24). ELISA indicated that MDA-MB-231 cells treated with Shh had significantly enhanced MMP-9 and MMP-2 levels. By contrast, inhibition of the Shh-Gli signaling pathway with cyclopamine led to significantly reduced MMP-9 and MMP-2 levels (Fig. 4B). Cell invasion capacity was determined using a scratch assay, which revealed that pretreatment with Shh enhanced, while cyclopamine treatment reduced, MDA-MB-231 cell invasion (Fig. 4C). However pretreatment with cyclopamine prior to Shh stimulation significantly reduced Shh-induced cell invasion (Fig. 4C). These findings indicate that Shh-Gli activation enhances the EMT process and increases MMP-9/MMP-2 secretion, thereby promoting cell migration and invasion in breast cancer cells.